Protracted β-cell depolarization reduces surface expression of Ca_V1.2 and voltage-gated Ca²⁺ currents.

<p>A) Immunostaining of Ca_V1.2 and plasma membrane (PM) marker Na⁺/K⁺ ATPase in insulin-secreting INS-1 832/13 cells, before and after 30 min exposure to either 20 mM glucose or 70 mM KCl. B) Depicts the calculation of the ratio between the mean intensity of plasma membrane (I_PM) to that of the cytosol (I_cytosol). The PM area was defined by Na⁺/K⁺ ATPase staining. C) Average ratios of Ca_V1.2 expression under the conditions in A, n = 12. ** p<0.01 (ANOVA, F-test). D) Immunostaining of Ca_V1.2 and the early endosome marker EEA-1 before and after stimulation with 70 mM K⁺ or 20 mM glucose. E) Colocalization evaluated by Pearson’s correlation coefficient analysis which demonstrated colocalization of Ca_V1.2 and EEA-1 in the area “3″ whereas “1″ or “2″ represent no colocalization. F) Average colocalization of Ca_V1.2 to EEA-1 calculated under conditions as in (d), n = 10. * p<0.05, ** p<0.01 (ANOVA, F-test). G) Immunostaining of Ca_V1.2 and the recycling endosome marker Rab11 before and after 20 mM glucose stimulation. H) Average Ca_V1.2 colocalization to Rab11 detected under conditions as in G, n = 9. ** p<0.01. I) Representative Ca²⁺ currents measured by patch clamp under glucose stimulation. J) Average integrated whole-cell Ca²⁺ current (Q) in INS-1 832/13 cells before (n = 5 cells), during (n = 4 cells) and after (n = 4 cells) elevation of extracellular glucose from 5 to 20 mM. K) Immunoblotting of Ca_V1.2 in plasma membrane (PM), cytosol (C) and homogenate (H) fractions before and after 20 mM glucose stimulation. Scale bar in all images 5 µm.